Single molecule kinetics in familial hypertrophic cardiomyopathy transgenic heart

Cardiovascular diseases are the predominant cause of death in the United States and most of the European countries. Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death (SCD) at a young age. Familial hypertrophic cardiomyopathy (FHC) is a heritable form of cardiac hypertrophy caused by single-point mutations in genes encoding sarcomeric proteins including the ventricular myosin regulatory light chain (RLC). Among other mechanisms, FHC mutations are believed to alter the kinetics of the interaction between actin and myosin resulting in inefficient energy utilization and compromised function of the heart. In this study, we report direct measurements of actomyosin kinetics at single molecule level. Single molecule kinetics of transgenic (Tg) mouse hearts of Wild type and three different RLC mutations, D166V, R58Q and A13T are studied by Fluorescence Correlation Spectroscopy (FCS). D166V and R58Q have been associated with malignant FHC phenotype and sudden cardiac death where as A13T has been associated with a particular subtype of FHC defined by mid-left ventricular obstruction. The approach was based on the fact that during muscle contraction myosin cross-bridge delivers force impulse to actin which leads change in actin orientation. The orientational changes were followed by polarization of fluorescence. Orthogonal intensities were obtained by measuring components of polarized fluorescence of rhodamine dipole attached to actin. This technique was first optimized in skeletal muscle to determine the kinetics of contraction and then applied to FHC muscle. The conclusions drawn here may well result in improved risk assessment and ultimately may lead to development of cardiomyocyte and myosin light chain specific treatments.